Clostridium pasteurianum rubredoxin (Rd) is a paramagnetic protein containing a single iron atom. It has been overproduced in E. coli by using a T7 promoter/T7 polymerase system. The yield is about 18 mg/L of culture. This protein will be labeled uniformly with 13C and 15N. NMR spectroscopy will be used to study the isotopic enrichment of native rubredoxin and metal-substituted rubredoxins. The hyperfine-shifted resonances arising from four iron-ligated cysteines will be assigned by means of protein labeled selectively with isotopically labeled cysteines. 3D NMR solution structure of this protein will be constructed using sequence-specific assignments of NMR resonances of native C. pasteurianum Rd with hyperthermostable Pyrococcus furiosus Rd (Blake, P.R., Park, J.-B., Zhou, Z.H., Hare, D.R., Adams, M.W.W. and Summers M., Protein Sci. 1, 1508-1521, 1992). The factors which cause the high stability of P. furiosus Rd at high temperature will be examined. The complete sequence-specific assignments of 13C, 15N, and 1H NMR resonances of C. pasteurianum Rd and its diamagnetic metal-substituted analogs will help us to understand the mechanism of hyperfine-shifts.